Synergistic compositions for the selective control of tumor tissue

ABSTRACT

According to the invention, compositions are made available which have a strong cytotoxic effect which is largely selective on tumor tissue. The invention is based on the fact that certain benzoic acid derivatives have a strong synergistic effect as a mixture and destroy cancer cells selectively in a pH range of 7 or below, such as from 6.5 to 7.

FIELD OF THE INVENTION

[0001] This invention is a Divisional of U.S. patent application Ser.No. 09/413,449 filed on Nov. 15, 2000 which is, in turn, aContinuation-In-Part of U.S. application Ser. No. 09/446,570 filed onMar. 27, 2000, now U.S. Pat. No. 6,395,720. The invention relates tonovel synergistic compositions which selectively control tumor tissue,while healthy tissue is virtually unattacked. The novel compositions aretherefore outstandingly suitable for cancer therapy.

BACKGROUND OF THE INVENTION

[0002] Medicaments according to the prior art which are employed inchemotherapy as a rule only achieve partial success, i.e. they do notlead to a final cure. Moreover, the substances employed in the prior artfrequently act only in a certain tumor category. A further disadvantageof the currently known chemotherapeutics are their often harmful sideeffects, as chemotherapeutics can generally have a cytostatic effect onproliferating tissue. The known chemotherapeutics are alsounsatisfactory in the control of metastasis formation, and this is oneof the main reasons which has until now prevented a decisive outcome incancer therapy.

[0003] The fact that tumor tissue in an extracellular medium has alowered average pH of about 6.5 to 7.0 and the pH can even fall to 5 onthe cancer cell surface, while the pH in the normal tissue and in theblood is approximately 7.2 to 7.5, is known and described, for examplein DE-A 44 07 484 and in Tumor Biol., 1994, 15: 304-310. Thus each typeof tumor has an intrinsic average intercellular pH, which, for example,in the case of breast tumors is about 6.7 and in the case of colontumors about 6.9.

[0004] In the abovementioned publications, it is disclosed that due tothe lowering of the pH range in tumor cells the natural immune defenseis blocked, as the body's own defense cells react to cancer target cellswith full cytotoxicity only in a slightly basic medium of pH more than7. DE-A 44 07 484 therefore proposes to bring the acidic external mediumof cancer cells to the normal physiological pH level of 7 to 7.5 andthereby to control the cancer cells by means of the body's own immunedefense. For this purpose, the acidic external medium of cancer cells isbrought to a physiological pH of 7 to 7.5 either by artificialbasification measures or by the prevention of the acidification processitself.

[0005] The medicaments described in DE-A 44 07 484 admittedly representan advance in cancer therapy, it would be desirable, however, to haveavailable medicaments which besides the body's own immune defenseselectively control tumor cells and thus can be used as relativelylow-side-effect chemotherapeutics.

[0006] Accordingly, WO 96/30003 proposes to use those compounds for thecontrol of tumor tissue which, at a pH of less than 7, are protonated orrelease a substance, the protonated compound or the released substancehaving a more strongly destructive effect on cells than the unprotonatedcompound or the compound before release of the substance. For thesecompounds, WO 96/30003 discloses general formulae under which come amultiplicity of chemical compounds. Inter alia, 4-amino-2-hydroxybenzoicacid is mentioned as being effective there. Acetylsalicylic acid is alsomentioned as a possible active compound, but this compound is notpreferred.

[0007] The compounds of WO 96/30003 and also the mixtures generallyproposed there of two and more compounds admittedly do already have agood anti-tumor effect, but, as before, there is a need for medicamentswhich have an improved anti-tumor effect, in particular at pH values of7.0 or below, in particular in the range from 6.5 to 7.0.

[0008] It is therefore an object of the present invention to makeavailable novel medicaments which have a strong cytotoxic effect whichis largely selective on tumor tissue, in particular in a pH range fromapproximately 6.5 to approximately 7.0.

[0009] This object is solved by the subject of the patent claims.

[0010] The compositions according to the invention in principle act inthe same manner as the benzoic acid derivatives disclosed in WO96/30003. It has been shown, however, that these benzoic acidderivatives, which are known from WO 96/30003, do not have completelysatisfactory activity against tumor tissue. Surprisingly, however,individual benzoic acid derivatives as a mixture with one another have avery strongly synergistic action for the destruction of tumor tissue andin a pH range of approximately 7.0 and below cause a virtually completecell death in tumor tissue.

SUMMARY OF THE INVENTION

[0011] The compositions according to the invention are mixtures whichcontain at least two of the following benzoic acid derivatives orcinnamic acid derivatives, respectively, which act synergistically as amixture with one another:

[0012] 2-acetoxybenzoic acid (acetylsalicylic acid, Aspirin)

[0013] 2-hydroxybenzoic acid

[0014] 2-methoxybenzoic acid

[0015] 2,4-dihydroxybenzoic acid

[0016] 2,4-diacetoxybenzoic acid

[0017] 2-hydroxy-4-aminobenzoic acid

[0018] 2,4-dimethoxybenzoic acid

[0019] 2,6-dihydroxybenzoic acid

[0020] 2,6-dimethoxybenzoic acid

[0021] 2,3,4-trimethoxybenzoic acid

[0022] 2,4,6-trihydroxybenzoic acid

[0023] 2,4,6-trimethoxybenzoic acid

[0024] 5-(2,4-difluoro-phenyl)salicylic acid

[0025] α-cyano-3-hydroxycinnamic acid

[0026] α-cyano-4-hydroxycinnamic acid

[0027] α-fluorocinnamic acid

[0028] α-methylcinnamic acid and αα-acetamidocinnamic acid.

[0029] Preferably, the mixture contains at least two of the followingbenzoic acid derivatives, which act synergistically as a mixture withone another:

[0030] 2-acetoxybenzoic acid (acetylsalicylic acid, Aspirin)

[0031] 2-methoxybenzoic acid

[0032] 2,4-diacetoxybenzoic acid

[0033] 2-hydroxy-4-aminobenzoic acid

[0034] 2,4-dimethoxybenzoic acid

[0035] 2,6-dihydroxybenzoic acid

[0036] 2,6-dimethoxybenzoic acid

[0037] 2,3,4-trimethoxybenzoic acid

[0038] 2,4,6-trihydroxybenzoic acid and

[0039] 2,4,6-trimethoxybenzoic acid

[0040] These compounds are known as such, commercially available and canbe prepared by a person skilled in the art without problems. They showtheir particular therapeutic benefits, however, only in the compositionsaccording to the invention.

[0041] A synergistic effect is not seen with all mixtures of theabovementioned benzoic acid derivatives. Whether a synergistic actionmay be present in a mixture can be easily determined by a person skilledin the art taking into account the details below.

[0042] Synergistic compositions are, for example, the followingmixtures:

[0043] 2,6-dihydroxybenzoic acid/2-hydroxy-4-aminobenzoic acid

[0044] 2,6-dihydroxybenzoic acid/acetylsalicylic acid

[0045] 2,6-dihydroxybenzoic acid/2,4-diacetoxybenzoic acid

[0046] 2,6-dihydroxybenzoic acid/2,4-dimethoxybenzoic acid

[0047] 2-hydroxy-4-aminobenzoic acid/acetylsalicylic acid

[0048] 2-hydroxy-4-aminobenzoic acid/2,4-dimethoxybenzoic acid and

[0049] 2,4-dimethoxybenzoic acid/2-acetoxybenzoic acid.

[0050] Furthermore, the following mixtures can also be mentioned:

[0051] 2,4,6-trihydroxybenzoic acid/2,4-dimethoxybenzoic acid

[0052] 2,4,6-trihydroxybenzoic acid/2,6-dihydroxybenzoic acid

[0053] 2,4,6-trimethoxybenzoic acid/2,6-dihydroxybenzoic acid

[0054] 2,4,6-trimethoxybenzoic acid/2-hydroxy-4-aminobenzoic acid

[0055] 2,4,6-trimethoxybenzoic acid/2,4,6-trihydroxybenzoic acid

[0056] 2,4,6-trimethoxybenzoic acid/2,4-dimethoxybenzoic acid.

[0057] As further mixtures can also be mentioned:

[0058] 2-hydroxy-4-aminobenzoic acid/5-(2,4-difluorophenyl)-salicylicacid

[0059] 2-acetoxybenzoic acid/5-(2,4-difluorophenyl)salicylic acid

[0060] 2-acetoxybenzoic acid/α-cyano-3-hydroxycinnamic acid

[0061] 5-(2,4-difluorophenyl)salicylic acid/α-cyano-3-hydroxy-cinnamicacid and

[0062] 2-hydroxy-4-aminobenzoic acid/α-cyano-3-hydroxycinnamic acid.

[0063] The use of triple combinations of the mixtures is also especiallypreferred, whereby the following triple combinations have advantageoussynergistic properties:

[0064] 2-hydroxy-4-aminobenzoic acid/2-acetoxybenzoic acid15-(2,4-difluorophenyl)salicylic acid

[0065] 2-hydroxy-4-aminobenzoic acid 2-acetoxybenzoic acidα-cyano-3-hydroxycinnamic acid

[0066] 2-hydroxy-4-aminobenzoic acid/5-(2,4-difluorophenyl)-salicyclicacid/α-cyano-3-hydroxycinnamic acid and 2-acetoxybenzoicacid/5-(2,4-difluorophenyl)salicyclic acid/α-cyano-3-hydroxycinnamicacid.

[0067] In the abovementioned mixtures 2-hydroxybenzoic acid (salicyclicacid) can also be used instead of acetylsalicyclic acid.

[0068] An example which may be mentioned of a nonsynergistic compositionof the above constituents is a mixture of 2,4-diacetoxybenzoic acid and2-hydroxy-4-aminobenzoic acid.

[0069] On account of their pH sensitivity, the compositions according tothe invention are only activated in cancer tumors and metastatic areasand therefore represent an ideal cancer therapeutic. It is also to beparticularly emphasized that this novel cancer therapeutic actsgenerally on all tumor types independently of the specific type ofcancer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0070] FIGS. 1-4 show the photometric measurement relationship betweenthe initial pH and wavelength for the cells identified in Tables 1-3;

[0071] FIGS. 4-5 show a recording of the death of the cells in aELISA(apoptosis kit) with RT1 12 as a function of pH;

[0072]FIG. 6 is a graph of the activity of cellular SalicylicacidCombinations Against the Human Pancreas Carcinoma PAXF 736; and

[0073]FIG. 7 is a graph of Body Weight Changes During Treatment WithSalicylicacid Combinations in the Human Pancreas Carcinoma PAXF 736.

DETAILED DESCRIPTION OF THE INVENTION

[0074] It is assumed that the substance mixtures according to theinventionhave toxic properties in the protonated state and act ascellular toxins on the tumors.

[0075] It is known that the pHs in the extracellular tumor tissue can belowered again by about 0.5 pH units by inducing acidosis by glucoseadministration. Such an administration of glucose can likewise takeplace with the compositions according to the invention.

[0076] The compositions according to the invention can contain theactive compounds in any desired proportions provided that thesynergistic effect still occurs. How the activity of compounds and thusalso the presence of a synergistic effect can be detected is describedin detail, for example, in WO 96/30003, and in this respect referencecan be made to this publication. Reference is further made to thefollowing comparison examples.

[0077] The compositions according to the invention preferably containthe two active compounds in the ratio 1:9 to 9:1, particularlypreferably in the ration 2:1 to 1:2 and in particular in the ratio ofapproximately 1:1.

[0078] The synergistic activity of the compositions according to theinvention was clearly confirmed by in vitro experiments. In thefollowing, the following short forms are used for the followingsubstance names:

[0079] Substance 121=acetylsalicylic acid (2-acetoxybenzoic acid)

[0080] Substance 58a=2-hydroxy-4-aminobenzoic acid

[0081] Substance 132=2,4-diacetoxybenzoic acid

[0082] Substance 136=2,6-dihydroxybenzoic acid

[0083] Substance 188=2,4-dimethoxybenzoic acid

[0084] The experiments according to the invention were carried out asfollows:

[0085] The measurements were carried out using a “Cell Death DetectionELISA” kit, Cat. No. 1774 425 commercially available from Boehringer(Mannheim). The process instructions are additionally supplied byBoehringer.

[0086] The results can be seen from Tables 1 to 3 below. The OD valuecorresponds to the cell death. TABLE 1 (Photometric measurement after 35minutes) Compound Initial pH OD (405-490 nm) CAM (800 ng/ml) pH 6.00.818 pH 6.5 3.037 pH 7.0 2.725 pH 7.4 2.416 136 (5 mM) pH 6.0 0.187 pH6.5 0.918 pH 7.0 0.321 pH 7.4 0.382 136 (7 mM) pH 6.0 0.221 pH 6.5 1.287pH 7.0 0.524 pH 7.4 0.816 Negative control pH 6.0 0.642 pH 6.5 0.702 pH7.0 0.293 pH 7.4 0.268 Positive control 3.145

[0087] TABLE 2 (Photometric measurement after 35 minutes) CompoundInitial pH OD (405-490 nm) CAM (160 ng/ml) pH 6.0 0.344 pH 6.5 0.711 pH7.0 1.220 pH 7.4 1.923 58a (15 mM) pH 6.0 0.320 pH 6.5 0.282 pH 7.00.574 pH 7.4 1.835 121 (10 mM) pH 6.0 1.282 pH 6.5 0.163 pH 7.0 0.413 pH7.4 1.508 132 (10 mM) pH 6.0 2.856 pH 6.5 0.183 pH 7.0 0.088 pH 7.40.502 188 (15 mM) pH 6.0 0.256 pH 6.5 0.309 pH 7.0 0.502 pH 7.4 1.854Negative control pH 6.0 0.674 pH 6.5 0.648 pH 7.0 0.327 pH 7.4 0.322Positive control 1.940

[0088] TABLE 3 (Photometric measurement after 20 minutes) CompoundInitial pH OD (405-490 nm) CAM (800 ng/ml) pH 6.0 1.681 pH 6.5 1.708 pH7.0 1.791 pH 7.4 2.217 136 + 58a pH 6.0 0.206 (5 mM/10 mM) pH 6.5 0.745pH 7.0 2.036 pH 7.4 0.793 136 + 121 pH 6.0 0.750 (5 mM/5 mM) pH 6.50.486 pH 7.0 2.564 pH 7.4 0.970 136 + 132 pH 6.0 0.419 (5 mM/5 mM) pH6.5 0.328 1st experiment pH 7.0 0.802 pH 7.4 0.953 136 + 132 pH 6.00.640 (5 mM/5 mM) pH 6.5 0.274 2nd experiment pH 7.0 1.160 pH 7.4 1.124136 + 188 pH 6.0 0.211 (5 mM/10 mM) pH 6.5 1.728 pH 7.0 1.426 pH 7.40.804 58a + 121 pH 6.0 0.577 (10 mM/5 mM) pH 6.5 0.480 pH 7.0 2.227 pH7.4 1.238 58a + 132 pH 6.0 0.152 (10 mM/5 mM) pH 6.5 0.241 pH 7.0 0.417pH 7.4 1.082 58a + 188 pH 6.0 0.251 (10 mM/5 mM) pH 6.5 1.698 pH 7.02.939 pH 7.4 0.915 132 pH 6.0 0.407 (5 mM) pH 6.5 0.371 pH 7.0 0.422 pH7.4 0.916 Negative control pH 6.0 0.883 pH 6.5 0.794 pH 7.0 0.330 pH 7.40.579 Positive control 1.219

[0089] The results of the experiments are summarized in FIGS. 1 to 3,which correspond to Tables 1 to 3. In the figures, the cell death isrecorded in a ELISA (apoptosis kit) with RT112 as a function of the pH.That is to say the data on the Y axis of the figures is a measure of thecell death; the pH of the cells is indicated on the X axis. FIG. 1 andFIG. 2 show experiments in which individual compounds were employed.FIG. 3 shows the synergistic effect of the compositions according to theinvention compared with the individual compounds. It stands out that ata pH of between 6.5 and 7.0 the compositions according to the inventioncause a cell death which is preferably in the vicinity of or above theCAM value, which virtually corresponds to a complete destruction of thetumor tissue.

[0090] The apoptosis-inducing substance CAM used in the experiments iscamptothecin (CAM value). In the experiments, positive control meansthat instead of the apoptotic cancer cells induced by the substances ahistone-DNA complex with known detection sensitivity is employed fordetection, i.e. an artificial apoptosis product is provided. In theexperiments, negative control means that the same detection procedure iscarried out, but without addition of substances or substance mixtures.

[0091] From the comparison experiments it is obvious that certainmixtures of the abovementioned benzoic acid derivatives have asynergistic effect. The mixture of 2,4-diacetoxybenzoic acid and2-hydroxy-4-aminobenzoic acid does not show any synergistic effect, andthe ability of the mixture to destroy tumor cells is not higher thanthat of individual compounds in the example of 2,4-diacetoxybenzoicacid.

[0092] The compositions according to the invention can be formulated ina known manner for medicaments for mammals, preferably man. In themedicaments, the compositions according to the invention are present asa mixture with a pharmaceutical organic or inorganic excipient, which issuitable for enteral or parenteral administrations. The oraladministration of the compositions according to the invention by meansof tablets, capsules, powders or in liquid form, such as suspensions, insolution, as an emulsion or as a syrup, is particularly preferred.

[0093] In the case of formulation as tablets, customary pharmaceuticalexcipients such as sodium citrate, lactose, microcrystalline celluloseand starch, lubricants such as anhydrous silicic acid, hydrogenatedcastor oil, magnesium stearate, sodium lauryl sulfate and talc, as wellas binders such as starch paste, glucose, lactose, gum arabic, mannitol,magnesium trisilicate and talc are used. If the compositions accordingto the invention are to be administered by means of liquids, customaryliquid excipients can be used.

[0094] A formulation for injections and infusions is likewise preferred,as is known in the field and described in relevant standard works.

[0095] The compositions according to the invention can likewise beformulated in a manner known per se as depot formulations or to givemedicaments having delayed or sustained release.

[0096] The dosage form of the compositions according to the inventiondepends on the specific composition and further factors and can bedetermined by a person skilled in the art on the basis of the conditionof the patient to be treated, the severity and type of the disease to betreated, possible side effects of the substance mixtures administered,etc.

[0097] The dosage of the compositions according to the invention can bedetermined by a person skilled in the art depending on the specificdisease, the patient and other circumstances and is, for example, 50mg/kg of body weight up to 300 mg/kg of body weight, preferably 100mg/kg of body weight up to 200 mg/kg of body weight, of the compositionaccording to the invention per day.

[0098] It is obvious to the person skilled in the art that the compoundsof the compositions according to the invention can be administeredtogether or in succession at short time intervals such that they stillhave their synergistic effect. According to the invention, both thesimultaneous administration of a suitably formulated substance mixtureand the time-shifted or simultaneous administration of the suitablyformulated individual constituents of the compositions according to theinvention are included, provided that the time intervals between theadministration of the individual constituents are not so large that thesynergistic effect is lost.

[0099] The time interval between the administration of the individuallyformulated components is generally not more than 24 hours, preferablynot more than one hour. Especially preferred is the joinedadministration of the formulations or the administration of oneformulation immediately after the other.

[0100] Therefore, the invention also relates to a pharmaceutical packconsisting of two medicaments each of which contains at least onebenzoic acid derivative selected from

[0101] 2-acetoxybenzoic acid (acetylsalicylic acid, Aspirin)

[0102] 2-hydroxybenzoic acid

[0103] 2-methoxybenzoic acid

[0104] 2,4-dihydroxybenzoic acid

[0105] 2,4-diacetoxybenzoic acid

[0106] 2-hydroxy-4-aminobenzoic acid

[0107] 2,4-dimethoxybenzoic acid

[0108] 2,6-dihydroxybenzoic acid

[0109] 2,6-dimethoxybenzoic acid

[0110] 2,3,4-trimethoxybenzoic acid

[0111] 2,4,6-trihydroxybenzoic acid

[0112] 2,4,6-trimethoxybenzoic acid

[0113] 5-(2,4-difluoro-phenyl)salicylic acid

[0114] α-cyano-3-hydroxycinnamic acid

[0115] α-cyano-4-hydroxycinnamic acid

[0116] α-fluorocinnamic acid

[0117] α-methylcinnamic acid and

[0118] α-acetamidocinnamic acid.

[0119] and, if applicable, a pharmaceutically acceptable carrier orthinner for the joined or timely shifted administration, whereby thepharmaceutical pack contains at least two different benzoic acidderivatives.

[0120] The preferred combinations of active agents of the pharmaceuticalpacks correspond to the above-mentioned preferred mixtures of theinvention.

[0121] Pharmaceutical pack can also be defined as the hint, for exampleon the package insert of a benzoic acid containing preparation, thatthis benzoic acid containing preparation should be administered jointlyor timely shifted with another benzoic acid containing preparation. As aresult, the invention also relates to a pharmaceutical pack as definedabove, which is characterized in that the two medicaments are eachprovided in a separate package, whereby on one package insert of atleast one of the packages it is pointed to the joined or timely shiftedadministration with the other medicament.

[0122] The following galenic example illustrates the invention and isnot limiting.

EXAMPLE

[0123] A solution of 2-hydroxy-4-aminobenzoic acid in distilled water isprepared, whereby the concentration of the 2-hydroxy-4-aminobenzoic acidis selected in a way that the prepared solution is from isotonic (34mg/ml 2-hydroxy-4-aminobenzoic acid) to slightly hypertonic (48 mg/ml2-hydroxy-4-aminobenzoic acid), sterile and pyrogene free. Parallelthereto, a solution of 2-acetoxybenzoic acid obtainable under thetradename Aspisol is provided. Both solutions are administered to apatient intravenously one after the other, whereby the2-hydroxy-4-aminobenzoic acid is administered in a dosage of 300 mg/kgbody weight and the 2-acetoxybenzoic acid is administered in a dosage of50 mg/kg body weight.

[0124] In particular those compositions are highly active which contain5-(2,4-difluoro-phenyl)salicylic acid in combination with anothersalicylic compound and in particular with another salicylic compound asdefined earlier. Thus, particularly preferred compositions of thepresent invention are compositions which contain5-(2,4-difluoro-phenyl)salicylic acid in combination with one or morecompounds selected from the group consisting of

[0125] 2-acetoxybenzoic acid (acetylsalicylic acid)

[0126] 2-hydroxybenzoic acid

[0127] 2-methoxybenzoic acid

[0128] 2,4-dihydroxybenzoic acid

[0129] 2,4-diacetoxybenzoic acid

[0130] 2-hydroxy-4-aminobenzoic acid

[0131] 2,4-dimethoxybenzoic acid

[0132] 2,6-dihydroxybenzoic acid

[0133] 2,6-dimethoxybenzoic acid

[0134] 2,3,4-trimethoxybenzoic acid

[0135] 2,4,6-trihydroxybenzoic acid

[0136] 2,4,6-trimethoxybenzoic acid

[0137] salicylic acid.

[0138] Among the most preferred combinations of the present applicationare the following combinations:

[0139] 5-(2,4-difluoro-phenyl)salicylic acid→acetylsalicylic acid

[0140] 5-(2,4-difluoro-phenyl)salicylic acid→4-aminosalicylic acid

[0141] 5-(2,4-difluoro-phenyl)salicylic acid→2,6-dihydroxybenzoic acid

[0142] 5-(2,4-difluoro-phenyl)salicylic acid→2,4,6-trihydroxybenzoicacid

[0143] 5-(2,4-difluoro-phenyl)salicylic acid→4-aminosalicylic acid andacetalsalicylic acid.

[0144] To avoid any confusion in the following a short table ispresented summarizing the abbreviations and chemical names used in theFigures, the claims and the description:

[0145] Diflu=5-(2,4-difluoro-phenyl)salicylic acid (also known as“Diflunisal”)

[0146] ASA=acetylsalicylic acid=2-acetoxybenzoic acid

[0147] PAS=4-aminosalicylic acid=2-hydroxy-4-aminobenzoic acid

[0148] HSA=6-hydroxysalicylic acid=2,6-dihydroxybenzoic acid

[0149] DHSA=4,6-dihydroxysalicylic acid=2,4,6-trihydroxybenzoic acid.

[0150] The compositions of the present application show excellentactivity in tumor therapy and can be used for the treating of all kindsof tumor diseases. The activity has been shown by in vito and in vitroexperiments as explained below.

[0151] The antitumor activity of the following salicylate combinationsin vivo:

[0152] Diflu+PAS (4-Aminosalicylic acid)

[0153] Diflu+ASA (Acetyl-salicylic acid)

[0154] Diflu+PAS+ASA

[0155] Diflu=5-(2,4-Difluorophenyl)salicylic acid

[0156] has been tested in a pancreas human tumor xenograft model, i.e.,with human pancreatic tumors transplanted to nude mice. In this modelthe tumor growth can be observed as volume growth, because the tumorpieces of the pancreas carcinoma PAXF 736 are transplanted beneath theskin of the nude mice. The solutions of the salicylates were injectedinto the tail vain for 7 consecutive days. The compounds of thecombinations were administered at precise time points in order to obtainoptimal synergism and to respect plasma clearance times. The dosesapplied are: Diflu: 33 mg/kg/d PAS: 525 mg/kg/d  ASA: 72 mg/kg/d

[0157] in dual combinations and Diflu: 33 mg/kg/d PAS: 300 mg/kg/d  ASA:54 mg/kg/d

[0158] in the combination Diflu/PAS/ASA.

[0159] The control mice were injected physiological NaCl-solution. After7 days administration no further injections into the tail vain werepossible because of vain injuries.

[0160] Within the time of therapy the tumor growth is completelystopped. From there on the tumor starts slowly growing because ofincomplete killing of tumor cells in the tumor. The tumor volumes aremedium average volumes each taken from 8 tumors, i.e. from 4 mice eachbearing 2 tumors. The results are shown in FIG. 6.

[0161] The weight control of the mice during the therapy experiment isshown in FIG. 7. It means a control for toxic side effects. As noweight-loss is registered, no side effects by the administration ofthese salicylates are to be expected.

[0162] The anticancer activity of salicylate combinations can also beexamined by in vitro experiments with tumor cell lines. As a relevantcriterion of toxic effects on cancer cells the proliferation rate can beconsidered. The proliferation rate effected by salicylate combinatioinscan be measured in dependence of the extracellular pH-milieu. Theresults of such an experiment with the adherent tumor cell line RT112(bladder carcinoma) are shown in FIG. 4. (Details for cell cultures, seeMaterials & Methods below.)

[0163] The effect of salicylate combinations on the proliferation ofRT112 tumor cells is quantitated by the measurement of incorporatedtritiated thymidine ([3H]-TdR) into the cellular DNA of the cancer cellsby liquid scintillation counting (LSC). (Method details, see Material &Methods below.)

[0164] The experiment demonstrates that at pH<7.0 the proliferation ofthe tumor cells is stopped or markedly reduced by salicylatecombinations. But it also shows that monoadministration of Diflu, PAS,ASA, HSA and DHSA exhibits no effect or minor effect on theproliferation rate. Diflu/HSA shows comparable or even identicalefficiency as Diflu/DHSA does. Both these compound combinations reachthe best inhibition of all proliferation.

[0165] HSA 6-Hydroxysalicylic acid

[0166] DHSA=4,6-Dihydroxysalicylic acid.

[0167] The toxicity effect of salicylate combinations on cancer cellscan also be tested by experiments in which the function of themitochondrial respiration chain is examined. The respiratory chain rateis determined by the use of a calorimetric assay (XTT-assay) ofBoehringer Mannheim. In this assay the non-radioactive dye XTT {sodium3′-[1-(phenyl-amino-carbonyl)bis(4-methoxy-6-nitro) benzene sulfonicacid hydrate} is used to evaluate the influence of cytotoxic compounds.The mitochondrial “succinate-tetrazolium reductase” system cleaves thetetrazolium salt XTT to formazan. The amount of this dye is directlycorrelated fo the number of metabolically active cells, as the enzymesystem is only active in respiratory and viable cells (method details,see Material & Methods below). Formazan production has been measured asoptical density, and the results are shown in FIG. 5. The pH-dependentformazan production of the control is dramatically decreased by theapplication of the combinations of the present invention, such as

[0168] Diflu+PAS

[0169] Diflu+ASA

[0170] Diflu+DHSA.

[0171] The toxic activity starts at pH 7.2 and is completed at pH 6.9.

[0172] Details of the experiments are shown below.

[0173] Determination of Mitochondrial Activity of RT112 Using aColorimetric Assay (XTT-assay)

[0174] The non-radioactive, colorimetric assay system using XTT (sodium3′-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro)benzene sulfonic acid hydrate) (BOEHRINGER MANNHEIM) was used toevaluate the influence of cytotoxic molecules on the mitochondrialactivity of human tumor cells. The mitochondrial “succinate-tetrazoliumreductase” system cleaves the tetrazolium salt XTT to formazan. Theamount of this dye is directly correlated to the number of metabolicallyactive cells, as the enzyme system is only active in respirating andviable cells. For this purpose, RT112 cells were harvested, washed twiceand resuspended in culture media to yield a density of 2.5×10⁵/ml. 100μl/well of the RT112 cell suspension were seeded into 96 well microtiterplates (BECTON DICKINSON (BD), Heidelberg, FRG) and incubated over nightat 37° C. and 5% CO₂. The supernatant was replaced by 90 μl pH-adjustedculture medium (pH 6.0-7.4) and 10 μl of the solution of preferredsubstances or 100 μl/well pH-adjusted culture medium alone (control).The cells were incubated for various time periods at 37° C. and 5% CO₂.Thereafter, cells were washed twice and reincubated in fresh pH-adjustedculture medium. After the incubation period, pH of each well wasdetermined with highly sensitive microelectrodes before 50 μl XTTlabelling mixture was added. The cells were further incubated foradditional 4 h at 37° C. and 5% CO₂. The spectrophotometric absorbanceof the samples was measured using a microtiter plate (ELISA) reader(DYNATECH, Denkendorf, FRG) at a wavelength of 490 nm (630 nm asreference). Data acquisition and analysis was performed using theBioLinx 2.1 software (DYNATECH).

[0175] Materials and Methods

[0176] Culture of Tumor Cells

[0177] The human bladder tumor cell line. RT112 or the human chronicmyeloid leukemia cell line K562 were purchased from DKFZ Tumorzell—unddatenbank (Institut für experimentelle Pathologie, Heidelberg, FRG) orDSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,Braunschweig, FRG), respectively. The cells were cultured inHEPES-buffered RPMI 1640 (SIGMA, Deisenhofen, FRG) supplemented with 2mM L-glutamine (BIOCHROM, Berlin, FRG), 1% NEAE (BIOCHROM), 10%heat-inactivated fetal calf serum (BIOCHROM), 100 U/ml penicillin and 50μg/ml streptomycin (BIOCHROM) at 37° C. and 5% CO₂. Only mycoplasma-freecell cultures were used; this was frequently tested using a specificdetection kit (BOEHRINGER MANNHEIM, Mannheim, FRG).

[0178] [³H]-TdR Incorporation Assay

[0179] The influence of cytotoxic molecules on the proliferation ofRT112 tumor cells was quantitated by the measurement of the incorporatedtritiated thymidine ([³H]-TdR) into the cellular DNA of the tumor cellsby liquid scintillation counting (LSC). In brief, RT112 cells wereharvested, washed twice and resuspended in culture media (2.5×10⁵/ml).100 μl/well of the RT112 cell suspension were seeded into 96 wellmicrotiter plates (BECTON DICKINSON) and incubated over night at 37° C.and 5% CO₂. The supernatant was replaced by 90 μl pH-adjusted culturemedium (pH 6.0-7.4) and 10 μl of the solution of preferred substances or100 μl/well pH-adjusted culture medium alone (control). The cells wereincubated for various time periods at 37° C. and 5% CO₂. Thereafter,cells were washed twice, reincubated in fresh pH-adjusted culture mediumand pulsed for 24 h by the addition of 23.125 kBq [³H]TdR/well (925kBq/ml; AMERSHAM, Braunschweig, FRG). After freezing and thawing,radioactive DNA of the cultures were transferred to glass fiber filterswith an automatic cell harvester (PHARMACIA). The incorporatedradioactivity was measured in a liquid scintillation counter(PHARMACIA).

[0180] Preferably, the compositions of the present invention contain5-(2,4-difluoro-phenyl)salicylic acid and one other salicylic compoundor two other salicylic compounds. Thus the medicaments of the presentinvention contain preferably two or three pharmaceutically activeingredients.

1. A synergistic composition comprising a mixture of at least twobenzoic acid derivatives selected from 2-acetoxybenzoic acid(acetylsalicylic acid) 2-hydroxybenzoic acid 2-methoxybenzoic acid2,4-dihydroxybenzoic acid 2,4-diacetoxybenzoic acid2-hydroxy-4-aminobenzoic acid 2,4-dimethoxybenzoic acid2,6-dihydroxybenzoic acid 2,6-dimethoxybenzoic acid2,3,4-trimethoxybenzoic acid 2,4,6-trihydroxybenzoic acid2,4,6-trimethoxybenzoic acid 5-(2,4-difluoro-phenyl)salicylic acidα-cyano-3-hydroxycinnamic acid α-cyano-4-hydroxycinnamic acidα-fluorocinnamic acid α-methylcinnamic acid and α-acetamidocinnamicacid.
 2. A synergistic composition according to claim 1, characterizedin that it comprises a synergistic mixture of 2,6-dihydroxy and2-hydroxy-4-aminobenzoic acid, 2,6-dihydroxy and 2-acetoxybenzoic acid(acetylsalicylic acid), 2,6-dihydroxy and 2,4-diacetoxybenzoic acid,2,6-dihydroxy and 2,4-dimethoxybenzoic acid, 2-hydroxy-4-amino- and2-acetoxybenzoic acid (acetylsalicylic acid), 2-hydroxy-4-amino- and2,4-dimethoxybenzoic acid, 2,4-dimethoxy- and 2-acetoxybenzoic acid,2,4,6-trihydroxy- and 2,4-dimethoxybenzoic acid, 2,4,6-trihydroxy- and2,6-dihydroxybenzoic acid, 2,4,6-trimethoxy- and 2,6-dihydroxybenzoicacid, 2,4,6-trimethoxy- and 2-hydroxy-4-aminobenzoic acid,2,4,6-trimethoxy- and 2,4,6-trihydroxybenzoic acid or of2,4,6-trimethoxy- and 2,4-dimethoxybenzoic acid.
 3. A synergisticcomposition according to claim 1, characterized in that it comprises asynergistic mixture of 2-hydroxy-4-aminobenzoic acid and5-(2,4-difluorophenyl)-salicylic acid, 2-acetoxybenzoic acid and5-(2,4-difluorophenyl)salicylic acid, 2-acetoxybenzoic acid andα-cyano-3-hydroxycinnamic acid, 5-(2,4-difluorophenyl)salicylic acid andα-cyano-3-hydroxy-cinnamic acid, 2-hydroxy-4-aminobenzoic acid andα-cyano-3-hydroxycinnamic acid, 2-hydroxy-4-aminobenzoic acid and2-acetoxybenzoic acid and 5-(2,4-difluorophenyl)salicylic acid,2-hydroxy-4-aminobenzoic acid and 2-acetoxybenzoic acid andα-cyano-3-hydroxycinnamic acid, 2-hydroxy-4-aminobenzoic acid and5-(2,4-difluorophenyl)-salicylic acid and α-cyano-3-hydroxycinnamic acidor of 2-acetoxybenzoic acid and 5-(2,4-difluorophenyl)salicylic acid andα-cyano-3-hydroxycinnamic acid.
 4. The composition as claimed in claim 1comprising 2-hydroxy-4-aminobenzoic acid as a mixture with2,4-dimethoxybenzoic acid.
 5. The composition as claimed in claim 1comprising 2,6-dihydroxybenzoic acid as a mixture with acetylsalicylicacid.
 6. The composition as claimed in claim 1 comprising2-hydroxy-4-aminobenzoic acid as a mixture with acetylsalicylic acid. 7.The composition as claimed in claim 1 comprising2-hydroxy-4-aminobenzoic acid as a mixture with 2,6-dihydroxybenzoicacid.
 8. The composition as claimed in claim 1 comprising2,6-dihydroxybenzoic acid as a mixture with 2,4-dimethoxybenzoic acid.9. The composition as claimed in one of claims 1 to 8 for use as amedicament.
 10. The composition as claimed in one of claims 1 to 8 foruse as an antitumor agent.
 11. The medicament comprising a compositionas claimed in one of claims 1 to 8 and a pharmaceutically tolerableinert pharmaceutical excipient.
 12. The medicament as claimed in claim11, characterized in that it comprises the active agents in a ratio of1:9 to 9:1, preferably in a ratio of 2:1 to 1:2 and most preferably in aratio of 1:1.
 13. The medicament as claimed in claim 11 or 12,characterized in that it additionally comprises glucose.
 14. The use ofa composition as claimed in one of claims 1 to 8 for the preparation ofa medicament for the treatment of carcinomatous disorders.
 15. Apharmaceutical pack consisting of two medicaments each comprising atleast one benzoic acid derivative selected from 2-acetoxybenzoic acid(acetylsalicylic acid) 2-hydroxybenzoic acid 2-methoxybenzoic acid2,4-dihydroxybenzoic acid 2,4-diacetoxybenzoic acid2-hydroxy-4-aminobenzoic acid 2,4-dimethoxybenzoic acid2,6-dihydroxybenzoic acid 2,6-dimethoxybenzoic acid2,3,4-trimethoxybenzoic acid 2,4,6-trihydroxybenzoic acid2,4,6-trimethoxybenzoic acid 5-(2,4-difluoro-phenyl)salicylic acidα-cyano-3-hydroxycinnamic acid α-cyano-4-hydroxycinnamic acidα-fluorocinnamic acid α-methylcinnamic acid and α-acetamidocinnamic acidfor joined or timely shifted administration, wherein the pharmaceuticalpack comprises at least two different benzoic acid derivatives.
 16. Apharmaceutical pack according to claim 15, characterized in that the twomedicaments are provided in different packages, wherein on the packageinsert of one medicament and/or on the package insert of the othermedicament it is pointed to the joined or timely shifted administrationof the medicaments.
 17. Composition comprising5-(2,4-difluorophenyl)salicylic acid and at least one other salicylicacid derivative.
 18. Composition according to claim 17 wherein the atleast one other salicylic acid derivative is selected from the groupconsisting of 2-acetoxybenzoic acid (acetylsalicylic acid)2-hydroxybenzoic acid 2-methoxybenzoic acid 2,4-dihydroxybenzoic acid2,4-diacetoxybenzoic acid 2-hydroxy-4-aminobenzoic acid2,4-dimethoxybenzoic acid 2,6-dihydroxybenzoic acid 2,6-dimethoxybenzoicacid 2,3,4-trimethoxybenzoic acid 2,4,6-trihydroxybenzoic acid2,4,6-trimethoxybenzoic acid salicylic acid.
 19. Composition accordingto claim 18 wherein the salicylic acid derivative is selected from thegroup consisting of 2-hydroxy-4-aminobenzoic acid, 2-acetoxybenzoicacid, salicylic acid, 2,6-dihydroxybenzoic acid and2,4,6-trihydroxybenzoic acid.
 20. Composition according to claim 17comprising 5-(2,4-difluorophenyl)salicylic acid in combination with2-hydroxy-4-aminobenzoic acid.
 21. Composition according to claim 17comprising 5-(2,4-difluorophenyl)salicylic acid in combination with2-acetoxybenzoic acid.
 22. Composition according to claim 17 comprising5-(2,4-difluorophenyl)salicylic acid in combination with2,6-dihydroxybenzoic acid.
 23. Composition according to claim 17comprising 5-(2,4-difluorophenyl)salicylic acid in combination with2,4,6-trihydroxybenzoic acid.
 24. Composition according to claim 17comprising 5-(2,4-difluorophenyl)salicylic acid in combination withacetylsalicylic acid and 2-hydroxy-4-aminobenzoic acid.
 25. Medicamentcomprising a composition as claimed in claim 17 and a pharmaceuticallytolerable excipient.
 26. Medicament comprising a composition as claimedin claim 18 and a pharmaceutically tolerable excipient.
 27. Medicamentcomprising a composition as claimed in claim 19 and a pharmaceuticallytolerable excipient.
 28. Medicament comprising a composition as claimedin claim 20 and a pharmaceutically tolerable excipient.
 29. Medicamentcomprising a composition as claimed in claim 21 and a pharmaceuticallytolerable excipient.
 30. Medicament comprising a composition as claimedin claim 22 and a pharmaceutically tolerable excipient.
 31. Medicamentcomprising a composition as claimed in claim 23 and a pharmaceuticallytolerable excipient.
 32. Medicament comprising a composition as claimedin claim 24 and a pharmaceutically tolerable excipient. 33.Pharmaceutical pack consisting of two medicaments wherein one medicamentcomprises 5-(2,4-difluoro-phenyl)salicylic acid and the other medicamentcomprises at least one other salicylic acid derivative for joint ortimely shifted administration.
 34. Pharmaceutical pack according toclaim 33 wherein the at least one other salicylic acid derivative isselected from the group consisting of 2-acetoxybenzoic acid(acetylsalicylic acid) -2-hydroxybenzoic acid 2-methoxybenzoic acid2,4-dihydroxybenzoic acid 2,4-diacetoxybenzoic acid2-hydroxy-4-aminobenzoic acid 2,4-dimethoxybenzoic acid2,6-dihydroxybenzoic acid 2,6-dimethoxybenzoic acid2,3,4-trimethoxybenzoic acid 2,4,6-trihydroxybenzoic acid2,4,6-trimethoxybenzoic acid salicylic acid.
 35. Pharmaceutical packaccording to claim 33 wherein the at least one other salicylic acidderivative is selected from the group consisting of2-hydroxy-4-aminobenzoic acid, 2-acetoxybenzoic acid, salicylic acid,2,6-dihydroxybenzoic acid and 2,4,6-trihydroxybenzoic acid. 36.Pharmaceutical pack according to claim 33 wherein the at least one othersalicylic acid derivative is 2-hydroxy-4-aminobenzoic acid. 37.Pharmaceutical pack according to claim 33 wherein the at least one othersalicylic acid derivative is 2-acetoxybenzoic acid.
 38. Pharmaceuticalpack according to claim 33 wherein the at least one other salicylic acidderivative is 2,6-dihydroxybenzoic acid.
 39. Pharmaceutical packaccording to claim 33 wherein the at least one other salicylic acidderivative is 2,4,6-trihydroxybenzoic acid.
 40. Pharmaceutical packaccording to claim 33 wherein the other medicament contains acombination of acetylsalicylic acid and 2-hydroxy-4-aminobenzoic acid.41. Method of treating cancer wherein a composition of claim 17 isadministered to a patient in need of such a treatment.
 42. Method oftreating cancer wherein a composition of claim 18 is administered to apatient in need of such a treatment.
 43. Method of treating cancerwherein a composition of claim 19 is administered to a patient in needof such a treatment.
 44. Method of treating cancer wherein a compositionof claim 20 is administered to a patient in need of such a treatment.45. Method of treating cancer wherein a composition of claim 21 isadministered to a patient in need of such a treatment.
 46. Method oftreating cancer wherein a composition of claim 22 is administered to apatient in need of such a treatment.
 47. Method of treating cancerwherein a composition of claim 23 is administered to a patient in needof such a treatment.
 48. Method of treating cancer wherein a compositionof claim 24 is administered to a patient in need of such a treatment.